The insular cortex is one of the least understood regions of the brain due to a complex neuroanatomy involving granular, dysgranular, and agranular subregions with distinct connectivity. While several human imaging studies have implicated insula activity in fear conditioning and post-traumatic stress disorder (PTSD), few studies have attempted to investigate the role of the insula in rodents where higher resolution anatomical and functional techniques are possible. Previous rodent studies were mostly inconclusive in using blunt lesioning strategies to dissect the functional role of the insula in fer conditioning. Now, recent advances in viral transsynaptic tracing and optogenetics allow for the identification and experimental manipulation of discrete neural circuits for behavioral analysis. The proposed research plan applies the latest monosynaptic rabies viral tracing and optogenetic methods to determining the role of the insula in the fear learning process. As part of the Mouse Connectome Project in the lab of Dr. Hong-Wei Dong at USC, anterograde/retrograde tracer coinjections into the posterior, ventral, and dorsal agranular insular cortex (AIp, AIv, AId) have revealed that each subregion sends distinct patterns of input to the amygdala subnuclei involved in fear memory. Of these three insula subregions, the AIp provides the strongest input to the lateral part of the central nucleus of the amygdala (CEAl), whose neurons are critical for fear memory acquisition. Using a monosynaptic rabies viral tracing approach in mice, Aim 1 experiments will identify the neural circuits that provide direct input to CEA- projecting AIp neurons and establish the AIp as part of a structural neural network regulating the expression and memory of fear. Building upon this anatomical foundation, Aim 2 experiments under the mentorship of Drs. Li Zhang and Michael Fanselow will investigate the functional role of AIp projections to the CEA using excitatory and inhibitory optogenetics during behavioral fear conditioning and extinction paradigms. The results of these studies have important implications for our understanding of the neural circuits mediating fear learning and identifying a novel neuroanatomical target for PTSD treatment. In pursuit of these aims, the proposed training complements my strong neuroanatomical expertise with the ability to investigate the behavioral function of anatomically-defined neural circuits. Using the most advanced viral tracing, optogenetic, and behavioral techniques, the mentorship and guidance I will receive under this training grant is important for my personal goal of becoming an independent Principal Investigator studying structure/function relationships in neural networks.